1. Field of the Invention
The present invention relates, in general, to a motility stimulating peptide and compositions comprising the same. In particular, the present invention relates to a purified form of the peptide, for example, autotaxin (ATX); a DNA segment encoding autotaxin; recombinant DNA molecules containing the DNA segment; cells containing the recombinant DNA molecule; a method of producing autotaxin; antibodies to autotaxin; and methods of cancer diagnosis and therapy using the above referenced peptides and DNA segments.
2. Background Information
Cell motility plays an important role in embryonic events, adult tissue remodeling, wound healing, angiogenesis, immune defense, and metastasis of tumor cells (Singer, S. J. and Kupfer, A. (1986) Ann. Rev. Cell Biol. 2, 337-365). In normal physiologic processes, motility is tightly regulated. On the other hand, tumor cell motility may be aberrantly regulated or autoregulated. Tumor cells can respond in a motile fashion to a variety of agents. These include host-derived factors such as scatter factor (Rosen, E. M. et al., (1989) In Vitro Cell Devel. Biol. 25, 163-173) and growth factors (Kahan, B. W. et al., (1987) Cancer Res. 47, 6324-6328; Stracke, M. L. et al., Biochem. Biophys. Res. Comm. 153, 1076-1083; Tamm, I., et al., (1989) J. Exp. Med. 170, 1649-1669; Wang, J. M., et al. (1990) Biochem. Biophys. Res. Comm. 169, 165-170; and Jouanneau, J., et al. (1991) Proc. Natl. Acad. Sci. USA 88, 2893-2897), components of the extracellular matrix (McCarthy, J. B., et al. (1984) J. Cell Biol. 98, 1474-1480), and tumor-secreted or autocrine factors (Liotta, L. A., et al. (1988) Cancer Surveys 7, 631-652; Ruff, M., et al. (1985) Clin. Immunol. Immunopath. 37, 387-396; Atnip, K. D., et al. (1987) Biochem. Biophys. Res. Comm. 146, 996-1002; Ohnishi, T., et al. (1990) J. Neurosurg. 73, 881-888; Silletti, S., et al. (1991) Cancer Res. 51, 3507-3511.; and Watanabe, H., et al. (1991) J. Biol. Chem. 266, 13442-13448).
Many types of host-derived soluble factors act in a paracrine fashion to stimulate cell locomotion. Motility-stimulating proteins called "scatter factors" have been identified which are produced by embryonic fibroblasts and by smooth muscle cells (Stoker, M., et al. (1987) Nature 327, 239-242). Scatter factors stimulate random and directed motility by epithelial cells, keratinocytes, vascular endothelial cells and carcinoma cells (Stoker, M., et al. (1987) Nature 327, 239-242; Rosen, E. M., et al. (1990) Proc. Soc. Exp. Biol. Med. 195, 34-43; and Weidner, K. M., et al. (1990) J. Cell. Biol. 111, 2097-2108), but not fibroblasts. In addition, a number of host-secreted growth factors have been demonstrated to stimulate motility in tumor cells, including nerve growth factor (Kahan, B. W., et al. (1987) Cancer Res. 47, 6324-6328) insulin-like growth factor-I (Stracke, M. L., et al. (1988) Biochem. Biophys. Res. Comm. 153, 1076-1083), interleukin-6 (Tamm, I., et al. (1989) J. Exp. Med. 170, 1649-1669), interleukin-8 (Wang, J. M., et al. (1990) Biochem. Biophys. Res. Comm. 169, 165-170), and acidic fibroblast growth factor (Jouanneau, J., et al. (1991) Proc. Natl. Acad. Sci. USA 88, 2893-2897). These paracrine factors may influence "homing" or the directionality of tumor cell motility.
In contrast to these host-derived factors, many types of tumor cells have been found to produce proteins termed "autocrine motility factors" which stimulate motility by the same tumor cells which make the factor (Liotta, L. A., et al. (1986) Proc. Natl. Acad. Sci. USA 83, 3302-3306). Autocrine motility factors are not specific for a given type of cancer cell but have a wide spectrum of activity on many types of cancer cells (Kohn, E. C., et al. (1990) Int. J. Cancer 46, 287-292), with little effect on normal fibroblasts or leukocytes.
Autocrine motility factors identified to date act through cell-surface receptors (Stracke, M. L., et al. (1987) Biochem. Biophys. Res. Comm. 147, 339-345; Nabi, I. R., et al. (1990) Cancer Res. 50, 409-414; Watanabe, H., et al. (1991) J. Biol. Chem. 266, 13442-13448) resulting in pseudopodial protrusion (Guirguis, R., et al. (1987) Nature 329, 261-263) leading to both random and directed migration (Liotta, L. A., et al. (1986) Proc. Natl. Acad. Sci. USA 83, 3302-3306; Atnip, K. D., et al. (1987) Biochem. Biophys. Res. Comm. 146, 996-1002; Ohnishi, T., et al. (1990) J. Neurosurg. 73,881-888).
Prior studies of human A2058 melanoma cells have demonstrated that these cells are a particularly rich source of autocrine motility factors. An autocrine motility factor with a molecular mass of approximately 60 kDa has been previously isolated from the conditioned media of these cells. (Liotta, L. A., et al. (1986) Proc. Natl. Acad. Sci. USA 83, 3302-3306). Similar tumor cell derived or induced factors with the same molecular weight have subsequently been reported and purified by several investigators (Atnip, K. D., et al. (1987) Biochem. Biophys. Res. Comm. 146, 996-1002; Schnor, S. L., et al. (1988) J. Cell Sci. 90, 391-399; Ohnishi, T., et al. (1990) J. Neurosurg. 73, 881-888; Siletti, S., et al. (1991) Cancer Res. 51, 3507-3511; Watanabe, H., et al. (1990) J. Cell Biol. 111, 2097-2108). Such factors are thought to play a key role in tumor cell invasion.
Most of the motility factors identified to date have not been purified to homogeneity and have not been sequenced. The novel tumor motility factor of the present invention, named herein as autotaxin (ATX), has been purified and verified to be a homogeneous sample by 2-D gel electrophoresis. The protein of the present invention is unique from any previously identified or purified motility factor. The molecular size of ATX is about 125 kDa and it has an isoelectric point of approximately 7.7. ATX stimulates both random and directed migration of human A2058 melanoma cells at pM concentrations. The activity of this factor (ATX) is completely sensitive to inhibition by pertussis toxin. No significant homology has been found to exist between the protein of the invention and any mammalian protein including previous factors known to stimulate cell motility.
There is a great clinical need to predict the aggressiveness of a patient's individual tumor, to predict the local recurrence of treated tumors and to identify patients at high risk for development of invasive tumors. The present invention provides a functional marker which is functionally related to the invasive potential of human cancer. The invention further provides an assay for this secreted marker in body fluids, or in tissues. The assay of the invention can be used in the detection, diagnosis, and treatment of human malignancies and other inflamatory, fibrotic, infectious or healing disorders.